Mesenchymal stem cell differentiation

ABSTRACT

The present invention provides for methods and compositions for treating or preventing arthritis and joint injury.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA) represents the most common musculoskeletal disorder.Approximately 40 million Americans are currently affected and thisnumber is predicted to increase to 60 million within the next twentyyears as a result of the aging population and an increase in lifeexpectancy, making it the fourth leading cause of disability. OA ischaracterized by a slow degenerative breakdown of the joint includingboth the articular cartilage (containing the cells and matrix whichproduce lubrication and cushioning for the joint) and the subchondralbone underlying the articular cartilage. Current OA therapies includepain relief with oral NSAIDs or selective cyclooxygenase 2 (COX-2)inhibitors, intra-articular (IA) injection with agents such ascorticorsteroids and hyaluronan, and surgical approaches.

Mesenchymal stem cells (MSCs) are present in adult articular cartilageand upon isolation can be programmed in vitro to undergo differentiationto chondrocytes and other mesenchymal cell lineages. In part it isregulated by growth factors (TGFβs, BMPs), serum conditions andcell-cell contact.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of ameliorating or preventingarthritis or joint injury in a mammal. In some embodiments, the methodcomprises administering to a joint of the mammal a compositioncomprising an effective amount of a polypeptide comprising an amino acidsequence having at least 95% identity to SEQ ID NO:1 or 25, therebyameliorating or preventing arthritis or joint injury in the mammal.

In some embodiments, the individual has arthritis or joint injury. Insome embodiments, the individual does not have, but is at risk for,arthritis or joint injury.

In some embodiments, the polypeptide comprises SEQ ID NO:1 or 25.

In some embodiments, the amino acid sequence has at least 95% identityto SEQ ID NOs 2, 3, 4, 26, 27, or 28. In some embodiments, the aminoacid sequence comprises SEQ ID NOs: 2, 3, 4, 26, 27, or 28.

In some embodiments, the amino acid sequence is at least 80% identicalto any of SEQ ID NOs: 5-24. In some embodiments, the amino acid sequencecomprises any of SEQ ID NOs: 5-24.

In some embodiments, the arthritis is selected from the group consistingof osteoarthritis, traumatic arthritis, and autoimmune arthritis.

In some embodiments, the mammal is a human.

In some embodiments, the composition further comprises hyaluronic acid.

The present invention also provides methods of inducing differentiationof mesenchymal stem cells into chondrocytes which form the cartilagematrix. In some embodiments, the method comprises contacting mesenchymalstem cells with a sufficient amount of a polypeptide comprising an aminoacid sequence having at least 95% identity to SEQ ID NO:1 or 25 toinduce differentiation of the stem cells into chondrocytes.

In some embodiments, the method is performed in vitro. In someembodiments, the method is performed in vivo and the stem cells arepresent in a mammal. In some embodiments, the mammal is a human.

In some embodiments, the polypeptide comprises SEQ ID NO:1 or 25.

In some embodiments, the amino acid sequence has at least 95% identityto SEQ ID NOs: 2, 3, 4, 26, 27, or 28. In some embodiments, the aminoacid sequence comprises SEQ ID NOs: 2, 3, 4, 26, 27, or 28.

In some embodiments, the amino acid sequence is at least 80% identicalto any of SEQ ID NOs: 5-24. In some embodiments, the amino acid sequencecomprises any of SEQ ID NOs: 5-24.

The present invention also provides pharmaceutical compositions forintra-articular delivery and systemic delivery. In some embodiments, thecomposition comprising a pharmaceutically effective amount of apolypeptide comprising an amino acid sequence having at least 95%identity to SEQ ID NO:1 or 25.

In some embodiments, the composition further comprises hyaluronic acid.

In some embodiments, the polypeptide comprises SEQ ID NO:1 or 25.

In some embodiments, the amino acid sequence has at least 95% identityto SEQ ID NOs 2, 3, 4, 26, 27, or 28. In some embodiments, the aminoacid sequence comprises SEQ ID NOs:2, 3, 4, 26, 27, or 28.

In some embodiments, the amino acid sequence is at least 80% identicalto any of SEQ ID NOs: 5-24. In some embodiments, the amino acid sequencecomprises any of SEQ ID NOs: 5-24.

Additional features, advantages, and embodiments of the invention may beset forth or apparent from consideration of the following detaileddescription, and claims. Moreover, it is to be understood that both theforegoing summary of the invention and the following detaileddescription are exemplary and intended to provide further explanationwithout limiting the scope of the invention as claimed.

Definitions

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that has substantially the same structural and functionalcharacteristics of a naturally or non-naturally occurring polypeptide(e.g., ANGPTL3). Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res.15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al.J. Med. Chem. 30:1229 (1987), which are incorporated herein byreference). Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity), such as found in apolypeptide of interest, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of,e.g., —CH2NH—, —CH2S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-,—CH(OH)CH2-, and —CH2SO—. The mimetic can be either entirely composed ofsynthetic, non-natural analogues of amino acids, or, is a chimericmolecule of partly natural peptide amino acids and partly non-naturalanalogs of amino acids. The mimetic can also incorporate any amount ofnatural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. For example, a mimetic composition is within the scopeof the invention if it is capable of carrying out at least one activityof a polypeptide of interest.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Naturally encoded amino acids arethe 20 common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins    (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (e.g., a polypeptide of the invention), which doesnot comprise additions or deletions, for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity overa specified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. The invention provides polypeptides that are substantiallyidentical to the polypeptides, respectively, exemplified herein (e.g.,any of SEQ ID NOs: 1-28), as well as uses thereof including but nolimited to use for treating or preventing arthritis or joint injury.Optionally, the identity exists over a region that is at least about 50nucleotides in length, or more preferably over a region that is 100 to500 or 1000 or more nucleotides in length, or the entire length of thereference sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “hyaluronic acid” are used herein to include derivatives ofhyaluronic acid that include esters of hyaluronic acid, salts ofhyaluronic acid and also includes the term hyaluronan. The designationalso includes both low and high molecular weight forms of hyaluronansand crosslinked hyaluronans or hylans. Examples of such hyaluronans areSynvisc™ (Genzyme Corp. Cambridge, Mass.), ORTHOVISC™ (AnikaTherapeutics, Woburn, Mass.), and HYALGAN™ (Sanofi-Synthelabo Inc.,Malvern, Pa.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Quantitative assessment of MSC-induced type II collagenproduction during in vitro chondrogenesis. hMSCs (10,000/384 well) wereplated for 24 hours in hMSC growth media (Millipore). The cells weretreated with the proteins above for an additional 72 hours. The mediawas replaced with serum free DMEM and cultured an additional 14 days inthe absence of any additional stimuli. Upon termination, the cells werefixed with formalin, washed, stained with an antibody for type IIcollagen and counterstained with DAPI. The amount of type II collagenstaining was quantitated by high content imaging (Opera, Perkin Elmer).Data is pooled from 3 experiments in duplicate for each dose (n=6).

Characterization of ANGTPL3-induced chondrogenesis. FIG. 2A. hMSCs weregrown in pellet culture (1×106 cells/pellet) for 21 days in serum freeDMEM, 1× ITS and ANGTPL3 (where indicated). The media was replaced every3 days. Aggrecan mRNA expression was quantified using human Taqmanspecific probes according to the manufacturer's instructions (datapooled from 3 experiments in duplicate (n=6). FIG. 2B. Bovine cartilagewas isolated, punched into symmetric circles and put into organ culture.The slices were treated for 48 hours with 20 ng/ml TNFa and 10 ng/ml OSM(inflammatory mediators) to induce degradation of the cartilage matrixin the presence or absence of ANGPTL3 to identify the percent inhibitionof glycosaminoglycan release, an indicator of matrix damage (data pooledfrom 4 donors, n=12). FIG. 2C. Primary normal human chondrocytes andsynoviocytes (2500/384 well in growth media) were plated and culturedfor 24 hrs at 37° C. A moderate chondrocyte-specific cell growthincrease was demonstrated in response to ANGTPL3 over the24 hr period(data pooled from 2 experiments, 2 replicates/dose (n=4)).

mANGTPTL3. FIG. 3A. The primary protein structure and confirmedglycosylation sites. FIG. 3B. The atomic structure (1.8 angstroms) ofmANGTPL3 the C-terminal FLD (225-455).

Sequence alignment ANGPTL3 sequences. FIG. 4A. Sequence alignment of thehuman, mouse, bovine, and canine native ANGPTL3 proteins. FIG. 4B.Sequence alignment of the human, mouse, bovine, canine and equine nativeANGPTL3 proteins.

In vivo efficacy of mANGPTL3 in a surgical osteoarthritis model.Surgical transection of the anterior cruciate ligament (ACL), medialmeniscal tibial ligament (MMTL), and medial collateral ligament (MCL) ofthe right knee from C57BL/6 mice (n=12/group) was performed to induceinstability in the knee joint and thus lead to an OA phenotype. One weekfollowing surgery, the mice were dosed intra-articularly as indicatedonce/per for 3-4 weeks. FIG. 5A. Peripheral blood was collected on day28 following the surgery. The circulating type II collagen fragments(CTX-II) were quantified by an ELISA (Nordic biosciences). mANGPTL3dose=200 ng/knee, 3 weekly injections. FIG. 5B. Quantitative assessmentsof the tibial plateau were made on a 0-4 scale, 0 being normal and 5being severe OA (full thickness disruption of the cartilage). Twosections from each mouse were blindly graded by 2 observers. mANGPTL3dose=200 ng/knee, 3 weekly injections. FIG. 5C. OA pain was measured byincapacitance testing, or determining the percentage of time the mousestood on the surgical leg vs the other leg through the monitoringdevice. Readouts represent the pain response on day 36 after surgery (3doses weekly doses of ANGPTL3 at the indicated concentration).

DETAILED DESCRIPTION I. Introduction

The present invention is based, in part, on the discovery thatAngiopoietin-like 3 (ANGPTL3) stimulates chondrocyte differentiation inmesenchymal stern cells. Accordingly, the present invention provides formethods of induction of mesenchymal stem cell differentiation intochondrocytes. Further, the present invention provides for administrationof ANGPTL3 proteins to prevent or ameliorate arthritis or joint injuryby administrating an ANGPTL3 protein into a joint, the vertebrae,vertebral disc or systemically.

II. Angiopoietin-Like 3

Angiopoietin-like 3 is a member of the angiopoietin-like family ofsecreted factors. It is predominantly expressed in the liver, and hasthe characteristic structure of angiopoietins, consisting of a signalpeptide, N-terminal coiled-coil domain (CCD) and the C-terminalfibrinogen (FBN)-like domain. The FBN-like domain in angiopoietin-like 3was shown to bind αV/β3 integrins, and this binding induces endothelialcell adhesion and migration.

A variety of ANGPTL3 proteins can be used according to the presentinvention. As explained herein, native ANGPTL3 is generally cleaved invivo into amino-terminal and carboxyl terminal fragments. The presentinvention contemplates use of various ANGPTL3 proteins havingchondrogenic activity. In some embodiments, the invention provides foruse of full-length native (or variants thereof) ANGPTL3 protein aminoacid sequences. In some embodiments, the invention provides for ANGPTL3proteins comprising a portion (not the full-length native sequence) ofthe ANGPTL3 sequence, or a variant thereof, that retains chondrogenicactivity, i.e., not the amino-terminal end of the native protein. Insome embodiments, the ANGPTL3 proteins of the invention do not have theCCD domain and/or do not have significant CCD activity. Thus, in someembodiments, the ANGPTL3 proteins of the invention comprise at least afragment (e.g., at least 50, 100, 150, 200, 250 contiguous amino acids)of the native mouse (e.g., SEQ ID NO:12), human (e.g., SEQ ID NO:8),bovine (e.g., SEQ ID NO:16), dog (e.g., SEQ ID NO:20), or equine (e.g.SEQ ID NO:24) ANGPTL3 protein sequence or substantially identicalsequences, but do not comprise at least 200 contiguous amino-terminalamino acids of a native ANGPTL3 protein.

In some embodiments, the ANGPTL3 proteins of the invention comprise afibrinogen-like domain. In some embodiments, the ANGPTL3 proteins of theinvention comprise contiguous amino acids corresponding to amino acids207-455, 207-400, 207-350, 225-455, 225-400, 225-350, 241-455, 241-400,241-350 of the native mouse (e.g., SEQ ID NO:12), human (e.g., SEQ IDNO:8), bovine (e.g., SEQ ID NO:16), dog (e.g., SEQ ID NO:20), or equine(e.g. SEQ ID NO:24) ANGPTL3 protein sequence or are substantiallyidentical to such sequences, but do not include the flanking nativeANGPTL3 protein amino acid sequence. In some embodiments, the ANGPTL3proteins of the invention (including but not limited to any of SEQ IDNOs: 1-28) but lack at least a portion of the C-terminal sequence, e.g.,lack 10, 20, 30, 40, 50 amino acids from the C-terminus.

While the ANGPTL3 proteins of the invention as described above may notinclude native ANGPTL3 protein sequences flanking the regions describedabove, the ANGPTL3 proteins of the invention can include non-nativeANGPTL3 protein flanking sequences. For example, the chondrogenic activeportion of an ANGPTL3 protein can be fused to one or more heterologousamino acids to form a fusion protein. Fusion partner sequences caninclude, but are not limited to, amino acid tags, non-L (e.g., D-) aminoacids or other amino acid mimetics to extend in vivo half-life and/orprotease resistance, targeting sequences or other sequences.

The ANGPTL3 proteins of the invention encompass variants and truncationsof native ANGPTL3 proteins as well as variants and truncations of activefragments described herein. Active variants can be identified in anynumber of ways known to those of skill in the art. In some embodiments,amino acid alignments of active proteins can be established to identifythose positions that are invariant or that are include conserved aminoacid changes. SEQ ID NOs: 1, 2, 3, or 4 represent consensus sequencescomprising the invariant amino acids between certain areas (position241-455, 225-455 and 207-455, and native full-length, respectively) ofthe human, mouse, bovine, and canine native ANGPTL3 proteins. SEQ IDNOs: 25, 26, 27, or 28 represent consensus sequences comprising theinvariant amino acids between certain areas (position 241-455, 225-455and 207-455, and native full-length, respectively) of the human, mouse,bovine, canine, and equine native ANGPTL3 proteins. Thus, in someembodiments, the chondrogenic ANGPTL3 proteins of the invention compriseSEQ ID NOs: 1, 2, 3, 4, 25, 26, 27, or 28. In some embodiments, thechondrogenic ANGPTL3 proteins of the invention comprise an amino acidsequence substantially identical to any of SEQ ID NOs: 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 (asmeasured across the length of SEQ ID NOs).

The ANGPTL3 proteins of the invention have chondrogenic activity. Asdefined herein, chondrogenesis or chondrogenic activity refers to thedevelopment of chondrocytes from MSCs. Indicators of chondrogenicactivity include, but are not limited to, cartilage matrix production.Cartilage matrix production may be measured by various markers, forexample, such as Sox9, type II collagen, or glycosaminoglycan (GAG)production. In some embodiments, GAG production is measured as a markerfor cartilage matrix production. In some embodiments, a 3-fold increasein GAG production with cartilage specific protein expression indicatespositive cartilage matrix production.

In some embodiments, the ANGPTL3 polypeptides of the invention willcomprise at least one non-naturally encoded amino acid. Methods ofmaking and introducing a non-naturally-occurring amino acid into aprotein are known. See, e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647.The general principles for the production of orthogonal translationsystems that are suitable for making proteins that comprise one or moredesired unnatural amino acid are known in the art, as are the generalmethods for producing orthogonal translation systems. For example, seeInternational Publication Numbers WO 2002/086075, entitled “METHODS ANDCOMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNASYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THEEUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004; WO2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATIONCOMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS” and WO2007/103490, filed Mar. 7, 2007, entitled “SYSTEMS FOR THE EXPRESSION OFORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS.” Each ofthese applications is incorporated herein by reference in its entirety.For discussion of orthogonal translation systems that incorporateunnatural amino acids, and methods for their production and use, seealso, Wang and Schultz, (2005) “Expanding the Genetic Code.” AngewandteChemie Int Ed 44: 34-66; Xie and Schultz, (2005) “An Expanding GeneticCode.” Methods 36: 227-238; Xie and Schultz, (2005) “Adding Amino Acidsto the Genetic Repertoire.” Curr Opinion in Chemical Biology 9: 548-554;and Wang, et al., (2006) “Expanding the Genetic Code.” Annu Rev BiophysBiomol Struct 35: 225-249; Deiters, et al, (2005) “In vivo incorporationof an alkyne into proteins in Escherichia coli.” Bioorganic & MedicinalChemistry Letters 15:1521-1524; Chin, et al., (2002) “Addition ofp-Azido-L-phenylalanine to the Genetic Code of Escherichia coli.” J AmChem Soc 124: 9026-9027; and International Publication No.WO2006/034332, filed on Sep. 20, 2005, the contents of each of which areincorporated by reference in their entirety. Additional details arefound in U.S. Pat. No. 7,045,337; U.S. Pat. No. 7,083,970; U.S. Pat. No.7,238,510; U.S. Pat. No. 7,129,333; U.S. Pat. No. 7,262,040; U.S. Pat.No. 7,183,082; U.S. Pat. No. 7,199,222; and U.S. Pat. No. 7,217,809.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the common amino acids or pyrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

A non-naturally encoded amino acid is typically any structure having anysubstituent side chain other than one used in the twenty natural aminoacids. Because the non-naturally encoded amino acids of the inventiontypically differ from the natural amino acids only in the structure ofthe side chain, the non-naturally encoded amino acids form amide bondswith other amino acids, including but not limited to, natural ornon-naturally encoded, in the same manner in which they are formed innaturally occurring polypeptides. However, the non-naturally encodedamino acids have side chain groups that distinguish them from thenatural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Another type of modification that can optionally be introduced into theANGPTL3 proteins of the invention (e.g. within the polypeptide chain orat either the N- or C-terminal), e.g., to extend in vivo half-life, isPEGylation or incorporation of long-chain polyethylene glycol polymers(PEG). Introduction of PEG or long chain polymers of PEG increases theeffective molecular weight of the present polypeptides, for example, toprevent rapid filtration into the urine. In some embodiments, a Lysineresidue in the ANGPTL3 sequence is conjugated to PEG directly or througha linker. Such linker can be, for example, a Glu residue or an acylresidue containing a thiol functional group for linkage to theappropriately modified PEG chain. An alternative method for introducinga PEG chain is to first introduce a Cys residue at the C-terminus or atsolvent exposed residues such as replacements for Arg or Lys residues.This Cys residue is then site-specifically attached to a PEG chaincontaining, for example, a maleimide function. Methods for incorporatingPEG or long chain polymers of PEG are well known in the art (described,for example, in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8(2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev. 55: 217-50(2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76(2002)), the contents of which is incorporated herein by reference.Other methods of polymer conjugations known in the art can also be usedin the present invention. In some embodiments,poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) is introduced as apolymer conjugate with the ANGPTL3 proteins of the invention (see, e.g.,WO2008/098930; Lewis, et al., Bioconjug Chem., 19: 2144-55 (2008)). Insome embodiments, a phosphorylcholine-containing polymer conjugate withthe ANGPTL3 proteins can be used in the present invention. A person ofskill would readily recognize that other biocompatible polymerconjugates can be utilized.

A more recently reported alternative approach for incorporating PEG orPEG polymers through incorporation of non-natural amino acids (asdescribed above) can be performed with the present polypeptides. Thisapproach utilizes an evolved tRNA/tRNA synthetase pair and is coded inthe expression plasmid by the amber suppressor codon (Deiters, A, et al.(2004). Bio-org. Med. Chem. Lett. 14, 5743-5). For example,p-azidophenylalanine can be incorporated into the present polypeptidesand then reacted with a PEG polymer having an acetylene moiety in thepresence of a reducing agent and copper ions to facilitate an organicreaction known as “Huisgen [3+2]cycloaddition.”

In certain embodiments, the present invention contemplates specificmutations of the ANGPTL3 proteins so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, including but not limited to,O-linked or N-linked glycosylation sites. In certain embodiments, theANGPTL3 proteins of the present invention have glycosylation sites andpatterns unaltered relative to the naturally-occurring ANGPTL3 proteins.In certain embodiments, a variant of ANGPTL3 proteins includes aglycosylation variant wherein the number and/or type of glycosylationsites have been altered relative to the naturally-occurring ANGPTL3proteins. In certain embodiments, a variant of a polypeptide comprises agreater or a lesser number of N-linked glycosylation sites relative to anative polypeptide. An N-linked glycosylation site is characterized bythe sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. In certain embodiments, arearrangement of N-linked carbohydrate chains is provided, wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

Exemplary ANGPTL3 proteins variants include cysteine variants whereinone or more cysteine residues are deleted from or substituted foranother amino acid (e.g., serine) relative to the amino acid sequence ofthe naturally-occurring ANGPTL3 proteins. In certain embodiments,cysteine variants may be useful when ANGPTL3 proteins must be refoldedinto a biologically active conformation such as after the isolation ofinsoluble inclusion bodies. In certain embodiments, cysteine variantshave fewer cysteine residues than the native polypeptide. In certainembodiments, cysteine variants have an even number of cysteine residuesto minimize interactions resulting from unpaired cysteines.

In some embodiments, functional variants or modified forms of theANGPTL3 proteins include fusion proteins of an ANGPTL3 protein of theinvention and one or more fusion domains. Well known examples of fusiondomains include, but are not limited to, polyhistidine, Glu-Glu,glutathione S transferase (GST), thioredoxin, protein A, protein G, animmunoglobulin heavy chain constant region (Fc), maltose binding protein(MBP), or human serum albumin. A fusion domain may be selected so as toconfer a desired property. For example, some fusion domains areparticularly useful for isolation of the fusion proteins by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt-conjugated resins are used. Many of such matricesare available in “kit” form, such as the Pharmacia GST purificationsystem and the QLAexpress™ system (Qiagen) useful with (HIS₆) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the ANGPTL3 proteins. Examples of such detectiondomains include the various fluorescent proteins (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which aspecific antibody is available. Well known epitope tags for whichspecific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain embodiments, anANGPTL3 protein is fused with a domain that stabilizes the ANGPTL3protein in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, as desired).

III. Angiopoietin-Like 3 Proteins Disease Indications

It is contemplated that the polypeptides, compositions, and methods ofthe present invention may be used to treat or prevent any type ofarthritis or joint injury. It is further contemplated that thepolypeptides, compositions, and methods of the present invention may beused to treat or prevent various cartilagenous disorders. In someembodiments, the proteins of the invention are administered to preventarthritis or joint injury, for example where there is a genetic orfamily history of arthritis or joint injury or prior or during jointsurgery. Exemplary conditions or disorders to be treated or preventedwith the polypeptides, compositions, and methods of the invention,include, but are not limited to systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, osteoarthritis, degenerative discdisease, spondyloarthropathies, systemic sclerosis (scleroderma),idiopathic inflammatory myopathies (dermatomyositis, polymyositis),Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barr syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. In someembodiments of the invention, the polypeptides, compositions, andmethods of the present invention may be used to treat osteoarthritis.

In some embodiments, the polypeptides, compositions, and methods of thepresent invention provide a method for stimulating chondrocyteproliferation and cartilage production in cartilagenous tissues thathave been damaged due to traumatic injury or chondropathy. Of particularimportance for treatment are tissues that exhibit articulated surfaces,such as, spine, shoulder, elbow, wrist, joints of the fingers, hip,knee, ankle, and the joints of the feet. Examples of diseases that maybenefit from treatment include osteoarthritis, rheumatoid arthritis,other autoimmune diseases, or osteochondritis dessicans. In addition,cartilage malformation is often seen in forms of dwarfism in humanssuggesting that the polypeptides, compositions, and methods would beuseful in these patients.

It is contemplated that the polypeptides, compositions, and methods ofthe present invention may be used to treat a mammal. As used herein a“mammal” to any mammal classified as a mammal, including humans,domestic and farm animals, and zoo, sports or pet animals, such ascattle (e.g. cows), horses, dogs, sheep, pigs, rabbits, goats, cats,etc. In some embodiments of the invention, the mammal is a human.

In some embodiments, the polypeptides of the invention can beheterologous to the mammal to be treated. For example, a bovine ANGPTL3protein or fragments thereof, a protein or peptide derived from a bovineANGPTL3 protein (e.g., a modified bovine ANGPTL3 protein, a conservativevariant of bovine ANGPTL3 protein, a peptidomimetic derived from abovine ANGPTL3 protein) are used in the treatment of a human patient. Insome embodiments, a heterologous. ANGPTL3 protein can be used to expandchondrocyte populations in culture for transplantation. In someembodiments, the expanded cultures will then be admixed withpolypeptides and compositions homologous to the mammal to be treated,arid placed in the joint space or directly into the cartilage defect.Alternatively, the polypeptides of the invention are derived from thesame species, i.e., a human ANGPTL3 protein or fragments thereof, aprotein or peptide derived from a human ANGPTL3 protein (e.g., amodified human ANGPTL3 protein, a conservative variant of human ANGPTL3protein, a peptidomimetic derived from a human ANGPTL3 protein) is usedin the treatment of a human patient. By using a protein derived from thesame species of mammal as is being treated, one may avoid inadvertentimmune responses.

The polypeptides and compositions of the present invention can beapplied by direct injection into the synovial fluid of the joint,systemic administratin (oral or intravenously) or directly into thecartilage defect, either alone or complexed with a suitable carrier forextended release of protein. The polypeptides, compositions, and methodsof the present invention can also be used to expand chondrocytepopulations in culture for autogenous or allogenic chondrocytetransplantation. The transplantation can be optionally administered withconcurrent treatment consisting of administration of the polypeptidesand compositions of the present invention. In these procedures, forexample, chondrocytes can be harvested arthroscopically from anuninjured minor load-bearing area of the damaged joint, and can becultured in the presence of the polypeptides and compositions of thepresent invention to increase the number of cells prior totransplantation. The expanded cultures will then be admixed with thepolypeptides and compositions of the present invention, and placed inthe joint space or directly into the defect. The polypeptides andcompositions of the present invention can be used in combination withperiosteal or perichondrial grafts that contain cells that can formcartilage and/or help to hold the transplanted chondrocytes or theirprecursor cells in place. The polypeptides and compositions of thepresent invention can be used to repair cartilage damage in conjunctionwith lavage of the joint, stimulation of bone marrow, abrasionarthroplasty, subchondral drilling, or microfracture of the subchondralbone. Additionally, after the growth of cartilage due to theadministration of the polypeptides and compositions of the presentinvention, additional surgical treatment may be necessary to suitablycontour the newly formed cartilage surface.

IV. Pharmaceutical Compositions

The dose of a compound of the present invention for treating theabove-mentioned diseases or disorders varies depending upon the mannerof administration, the age and the body weight of the subject, and thecondition of the subject to be treated, and ultimately will be decidedby the attending physician or veterinarian. Such an amount of thecompound as determined by the attending physician or veterinarian isreferred to herein as an “effective amount.”

Formulations suitable for administration include excipients, includingbut not limited to, aqueous and non-aqueous solutions, isotonic sterilesolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular protein employed and the condition of the subject, as well asthe body weight or surface area of the area to be treated. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects that accompany the administration of aparticular protein or vector in a particular subject. Administration canbe accomplished via single or divided doses.

V. Methods of Administration

Any method for delivering the proteins of the invention to an affectedjoint can be used. In the practice of this invention, compositions canbe administered, for example, intra-articularly (i.e., into a joint),orally, intravenously. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.Solutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described. The proteins ofthe present invention can also be used effectively in combination withone or more additional active agents (e.g., hyaluronic acid or a saltthereof) depending on the desired therapy or effect.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 High Throughput Screen for Inducers of Chondrogenesis

To identify and define a new, non-invasive strategy for OA joint repair,we developed a non-biased high throughput screen of proteins capable ofselectively directing the differentiation of human mesenchymal stemcells (MSCs) to chondrocytes. The assay system models the resident MSCsin cartilage and identifies mediators that stimulate the natural repairpotential and enhance integrated cartilage regeneration. We choose totest a unique secreted protein library by a high throughput, cell-basedscreens of human and mouse MSCs. This approach provides a strategy thatallows the rapid identification of uncharacterized and native proteinligands that affect chondrogenesis.

To study these secreted proteins, two approaches were taken: theproduction of conditioned media (CM) from a mammalian producer cell line(HEK293T) and the generation of purified proteins from free style HEK-Fcells. Together these complementary approaches allowed for new targetidentification in a variety of applications.

Screening of proteins in the MSC assay identified the lead candidate,Angiopoeitin-like 3, abbreviated ANGPTL3. ANGPTL3 was identified in twoparallel but orthogonal initial screens of both the CM and purifiedproteins. In the proof of concept CM screen, C3H10t1/2 cells wereincubated for 7 days following transfer of the CM. Chondrogenesis wasfound to occur in wells containing ANGPTL3-75 assayed by Alcian bluestaining for detection of cartilage matrix production. ANGPTL3-75 wasindependently identified by screening 531 purified proteins forchondrocytic differentiation of human MSCs.

Following the initial screening assays, ANGPTL3 was subsequentlycharacterized in six secondary assays. These assays include: (1)monolayer culture of mouse mesenchymal C3H10t1/2 cells: induction oftype II collagen and Sox9 protein expression (markers of chondrogenesis)but no induction of osteocalcin (markers of osteogenesis); (2)inhibition of TNFα/oncostatin M (OSM)-induced nitric oxide (NO) releasein bovine chondrocytes; (3) inhibition of TNFα/OSM-inducedglycosaminoglycan (GAG) release in bovine cartilage organ culture; (4)induction of cartilage matrix gene expression (aggrecan) in a human MSCpellet culture system; (5) lack of toxicity in primary humanchondrocytes, human synovial fibroblasts, and human MSCs; (6)stimulation of proliferation of human chondrocytes.

The mouse ANGPTL3 protein functions in mouse, human and bovine celltypes and therefore was chosen for further characterization as itpotently induced (0.5-5 nM) chondrogenic differentiation although itshuman homolog retains a similar potency for chondrogenesis compared toother previously know inducers of chondrogenesis in the quantitativeimaged based type II collagen assay (FIG. 1). Both mouse and human MSCsdifferentiated in monolayer and pellet culture (respectively) for 18 to21 days when treated with 0.5-5 nM ANGPTL3 and express three cartilagespecific proteins: type II collagen, Sox9 and aggrecan. This wasassessed by both immunohistochemical staining and mRNA quantification byTaqman (FIG. 2A). Additionally, data suggest after culturing mouse MSCsfor 18 days, ANGPTL3 inhibits the spontaneous tendency towards afibrotic repair response through reduced expression of alkalinephosphatase. To assess the potential ability to prevent tissue damage,bovine cartilage organ cultures were stimulated with TNFα and oncostatinM (OSM). The stimulated glycosaminoglycan release, an indicator ofmatrix degeneration, was significantly inhibited by treatment withANGPTL3 (FIG. 2B). Additionally, treatment of primary human chondrocytesbut not human synoviocytes led to a 2 fold increase in cell growthwithin 24 hrs (FIG. 2C) suggesting a specificity of its action oncartilage. The protein also had no obvious in vitro toxic effects (<100μM) on the viability of in primary human chondrocytes, synovialfibroblasts and MSCs (data not shown).

In a direct comparison between the two candidates presently in clinicaltrials, treatment with 100 ng/ml of FGF18 or 100 ng/ml BMP7 could inducechondrogenic nodules, but with less overall matrix production comparedto ANGPTL3. FGF18 lacked the ability to increase Alcian Blue, Sox9 orType II collagen staining, indicating a lack of specificity of a truecartilage matrix. 100 ng/ml of BMP7 increased Alcian blue, Sox9 and TypeII collagen staining, but the significance was not as great at similarconcentrations.

Example 2 Expression of Recombinant Full Length ANGPTL3 and MutantProtein and Function Analysis

Mouse ANGPTL3 is predicted to be 51 kDa protein. It belongs to a familyof 7 identified Angiopoietin-like (ANGPTL) proteins that have structuralsimilarity to the angiopoiteins, but lack the ability to bind the Tie2receptor and have distinct functions. They contain an N-terminalcoiled-coil domain (CCD) and a C-terminal fibrinogen-like domain (FLD).ANGPTL proteins are tightly regulated by their microenvironment andinteractions with the extracellular matrix (ECM), yet the preciseinteraction sites nor partners have been elucidated in detail. ANGPTL3is secreted by the liver and circulates systemically. It is controlledthrough liver X receptors (LXR), with evidence that LXR-inducedhyper-triglyceridmedia is due to ANGPTL3 release. Interactions betweenthe CCD and the ECM through a putative heparin binding motif may lead toinhibition of cleavage at the proprotein convertase recognition sequence(R221-R224), similar to that reported for ANGPTL4. Cleavage results in asignificant increase in the CCD's ability to inhibit lipoprotein lipaseactivity (LPL), and thereby leads to increase of triglyceridemia (TG).This represents the major biological function of ANGPTL3 identifiedprior to the present invention. The C terminal FLD is sufficient toinduce endothelial cell adhesion and mediate angiogenesis after directimplantation in the rat cornea in vivo. It was also demonstrated thatrecombinant ANGPTL3 could bind to purified αVβ3 integrin and lead to anincrease in FAK, MAPK and AKT signaling in endothelial cells. These datasuggest the FLD interaction with integrins to mediate angiogenesis.

No expression of ANGPTL3 has been reported nor observed in our studiesusing western blotting in human chondrocytes, hMSCs or human synovialfibroblasts, and no expression of it was found in mouse knee joints.Furthermore, there are no reported activities for either fragment injoint cells relating to this novel chondrogenic function identified inour screen.

Our data indicates that full length ANGPTL3 is a novel mediator ofchondrogenesis and cartilage protection. We examined which domains ofANGPTL3 are critical for this novel chondrogenic function. A truncationseries of ANGPTL3 and full length protein have been expressed andpurified in an engineered HEK-S cell line that gives limited andhomogeneous glycosylation for detailed biophysical characterization.Mass spectrometric approaches confirmed the occupancy of all fourpredicted N-linked glycosylations (N115, N232, N296, and N357, FIG. 3A).From HEK endogenously processed fragments, the proteolytic cleavage sitebetween the CCD and FLD domain was determined to be R224. The fulllength ANGPTL3 elutes by size exclusion chromatography with a mass>400kDa, indicative of a trimer with heterogeneous glycosylation. The FLD(241-455), shown to be a monomer by size exclusion and static lightscattering, was crystallized. The atomic structure of the FLD wasdetermined at 1.8 angstroms resolution. The FLD structure revealed acore beta sheet configuration with helical stretches that orient threeloops toward the C-terminus, typical of other FLD homologs (FIG. 3B).The temperature factors in the C-terminal portion of the domain arehigher than the rest of the structure suggesting a high degree offlexibility in this region. Superposition with the ANGPTL2/Tie2structure (PDB code: 2GY7) suggests that the C-terminal portion of theFLD may be involved in protein-protein interactions.

The five truncation products generated have been evaluated in multiplechondrogenic assays. The results suggests no activity retention in theCCD alone, but activity remains in the FLD. Additionally, SMAD1phosphorylation is increased upon stimulation for 3 days with the fulllength or mutants G or H alone, suggesting activation of chondrogenicsignaling cascade.

Although systemic exposure of a protein injected intra-articularly islimited due to the synovial fluid lymph drainage, minimizing systemicexposure can sometimes be desirable. If there is systemic exposure, thecleavage of ANGPTL3 might lead to the release the CCD domain. Theincreased inhibition of LPL activity by CCD could lead to alteration ofthe patients' TG levels. Our results indicate potential clinicaladvantages of ANGPTL3. For instance, by dissecting where thechondrogenic activity in ANGPTL3 is localized we would minimize orexclude the unwanted systemic effects on lipid metabolism or theangiogenic properties. The chondrogenic activity is localized primarilyto the C terminus thus specific use of this domain would alleviate theconcerns of TG regulation of the full length molecule or N terminus. Asfull length ANGPTL3 is normally present in serum, there is no expectedimmunogenicity. An additional advantage of using the C terminus is thatit is cleaved and is a monomer (˜29 kDa) compared to the trimerized fulllength protein, thus possibly reducing systemic half life of any smallpercentage which might be present in circulation and limiting anypotential effect from angiogenesis.

Example 3 In Vivo Analysis of ANGPTL3

We have performed several in vivo evaluations of the full length ANGPTL3to address potential adverse events and intra-articular retention.Following intra-articular (IA) injection of the left knee joints of 8week old C57BL/10 mice with 3.6 μg of full length ANGPTL3, we evaluatedimmunohistochemically the expression in the presence and absence ofexposure for 24 hours. By 24 hours, very little to no detectable levelsof ANGPTL3 should remain in the synovial fluid as the typical turnoverby trans-synovial flow into the synovial lymph vessels for proteins andwater is approximately 2 hours. The results revealed no endogenousexpression of ANGPTL3 in untreated joints, but significant detection ofthe protein in the pericellular matrix in the articular cartilage and inthe menisci even at the 24 hour time point. Additionally no widespreadcytotoxicity to the chondrocytes or cartilage damage in vivo wasdetected. Following a series of 3 IA injections into the knee joints ofrats (once/week for three weeks), clinically there was no toxicity (nojoint swelling or alternations in gait) or evidence of an acuteinflammatory reaction in the joint of the rat. Histologically, there wasno increase in synovitis or uncontrolled proliferation in the joints ofthe five rats injected. As in the mouse, ANGPTL3 could be detected inthe cartilage matrix and surrounding the chondrocytes. These resultsindicate that ANGPTL3 does not cause any undesired effects to thecartilage itself, and that ANGPTL3 does enter into and is retained incartilage and menisci in vivo.

OA is not a single disease entity and can be considered the consequenceof various etiologic factors. In humans it is often caused by abnormalbiomechanical stress or genetic or acquired abnormalities of thearticular cartilage or bone. Therefore, choosing the “best” small animalOA model is difficult and multiple models should be explored todetermine the protective properties of any therapeutic. We havecompleted efficacy studies a chronic OA model (collagenase VII-inducedbased upon the research described by van der Kraan and colleagues) andan acute surgical model involving transection of the three of the majorligaments (ACL, MCL and MMTL) in the joint based upon the work ofGlasson et al. Both models induced pathological changes commonlyassociated with OA: loss of proteoglycan staining, erosion of thecartilage and bone, osteophyte formation and metaplastic alterations inthe synovium and ligaments can be evident 4-8 weeks after initiation ofOA. FIG. 5 depicts the regenerative capacity of ANGPTL3 surgical modelof OA. To begin to examine potential biomarkers for OA, peripheral bloodwas collected during the surgical model to measure the type II collagenfragments released due to cartilage damage (FIG. 5A). Histologicalanalysis and subsequent grading of the medial tibial plateau revealedregeneration in the cartilage matrix after treatment with 200 ngANGTPL3/knee once per week for 3 weeks (FIG. 5B).

In a 8 week surgical OA subset of mice, three doses of ANGPTL3 wereexamined for alleviation of OA-induced pain through incapacitancemeasurements. This method measures the weight distribution between thesurgical and non-surgical legs. On day 36 following surgery and 3 weeklytreatments with PRO1, as low as 100 ng/knee dosing demonstratedsignificant improvement compared to the vehicle treated surgical knees(FIG. 5C). These combined data provide concrete evidence that ANGTPL3has in vivo efficacy in two OA models (both pathological correction andpain diminishment) and supports the advancement of its development as anovel OA therapeutic.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTING SEQ ID NO: 1 241-455 consensusXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWTLIQHRXDGSQXFNETWENYXXGFGRLDGEFWLGLEKIYXIVXQSNYXLRXELXDWXD(X)KXYXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCPEXXSGGWWXXXXCGENNLNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX SEQ ID NO: 2 225-455 consensusTTPXXXXNEXXNXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWTLIQHRXDGSQXFNETWENYXXGFGRLDGEFWLGLEKIYXIVXQSNYXLRXELXDWXD(X)KXYXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCPEXXSGGWWXXXXCGENNLNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX SEQ ID NO: 3207-455 consensusXIXEXXEXSLSSKXRAPRTTPXXXXNEXXNXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWTLIQHRXDGSQXFNETWENYXXGFGRLDGEFWLGLEKIYXIVXQSNYXLRXELXDWXD(X)KXYXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCPEXXSGGWWXXXXCGENNLNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXX PXXSEQ ID NO: 4 Full length consensusMXTIKLXLXXXPLVIXSXXDXDXXSXDSXXXEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLXTXEIKEEEKELRRXTXXLQVKNEEVKNMSXELXSKXESLLEEKXXLQXKVXXLEXQLPXLIXXXXXXXEXXEVTSLKXXVEXQDNSIXXLLQXVEXQYXQLXQQXXQIKEIEXQLR(X)XXIXEXXEXSLSSKXRAPRTTPXXXXNEXXNXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWTLIQHRXDGSQXFNETWENYXXGFGRLDGEFWLGLEKIYXIVXQSNYXLRXELXDWXD(X)KXYXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCPEXXSGGWWXXXXCGENNLNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX(SEXXE) SEQ ID NO: 5 Human ANGPTL3 241-455GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGEGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD SEQ ID NO 6 Human ANGPTL3 225-455TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHENCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD SEQ ID NO 7Human ANGPTL3 207-455IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSEQ ID NO 8 Full length human ANGPTL3 gi|7656888|ref|NP_055310.1| [Homosapiens] MFTIKLLLFIVPLVISSRIDQDNSSEDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDEVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPERPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGEGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE SEQ ID NO: 9 Mouse ANGPTL3 241-455DLPADCSAVYNRGEHTSGVYTIKPRNSQGFNVYCDTQSGSPWTLIQHRKDGSQDFNETWENYEKGEGRLDGEFWLGLEKIYAIVQQSNYILRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMFSTWNHRAKGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRKLYAIKSSKMMLQPTT SEQ ID NO 10 Mouse ANGPTL3 225-455TTPPLQLNETENTEQDDLPADCSAVYNRGEHTSGVYTIKPRNSQGENVYCDTQSGSPWTLIQHRKDGSQDFNETWENYEKGFGRLDGEFWLGLEKIYAIVQQSNYILRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMESTWNHRAKGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRKLYAIKSSKMMLQPTT SEQ ID NO 11Mouse ANGPTL3 207-455IQEPSENSLSSKSRAPRTTPPLQLNETENTEQDDLPADCSAVYNRGEHTSGVYTIKPRNSQGFNVYCDTQSGSPWTLIQHRKDGSQDFNETWENYEKGFGRLDGEFWLGLEKIYAIVQQSNYILRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMFSTWNHRAKGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRKLYAIKSSKMMLQPTTSEQ ID NO 12 Full length mouse ANGPTL3 gi|33469117|ref|NP_038941.1| [Musmusculus] MHTIKLFLEVVPLVIASRVDPDLSSFDSAPSEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLRTNEIKEEEKELRRTTSTLQVKNEEVKNMSVELNSKLESLLEEKTALQHKVRALEEQLTNLILSPAGAQEHPEVTSLKSFVEQQDNSIRELLQSVEEQyKQLSQQHMQIKEIEKQLRKTGIQEPSENSLSSKSRAPRTTPPLQLNETENTEQDDLPADCSAVYNRGEHTSGVYTIKPRNSQGFNVYCDTQSGSPWTLIQHRKDGSQDFNETWENYEKGFGRLDGEFWLGLEKIYAIVQQSNYILRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMFSTWNHRAYGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRKLYAIKSSKMMLQPTT SEQ ID NO: 13 Bovine ANGPTL3 241-454DIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYILRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDHKAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRLYSIKATKMLIHPSD SEQ ID NO 14 Bovine ANGPTL3 225-454TTPSFHSNETKNVEHDDIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYILRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDHKAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRLYSIKATKMLIHPSD SEQ ID NO 15Bovine ANGPTL3 207-454IKESTEISLSSKPRAPRTTPSFHSNETKNVEHDDIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYILRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDHKAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRLYSIKATKMLIHPSDSEQ ID NO 16 Full length bovine ANGPTL3 gi|122692391|ref|NP_001073814.1|[Bos taurus]MYTIKLFLIIAPLVISSRTDQDYTSLDSISPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRATSKLQVKNEEVKNMSLELDSKLESLLEEKILLQQKVRYLEDQLTDLIKNQPQIQEYLEVTSLKTLVEQQDNSIKDLLQIVEEQYRQLNQQQSQIKEIENQLRRTGIKESTEISLSSKPRAPRTTPSFHSNETKNVEHDDIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYILRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDHKAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRLYSIKATKNILIHPSDSENSE SEQ ID NO: 17 Canine ANGPTL3 240-454DIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRLYSIKSTKMLIHPID SEQ ID NO: 18 Canine ANGPTL3 224-454TTPFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRLYSIKSTKMLIHPID SEQ ID NO: 19Canine ANGPTL3 206-454IQESTENSLSSKPRAPRTTPFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKEYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRLYSIKSTKMLIHPIDSEQ ID NO: 20 Full length canine ANGPTL3 gi|57086505|ref|XP_536686.1|[Canis familiaris]MYTIKLFLFIIPLVISSKIDRDYSSYDSVSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRTTSKLQVKNEEVKNMSLELNSKVESLLEEKILLQQKVRYLEKQLTSLIKNQPEIQEHPEVTSLKTFVEQQDNSIKDLLQTVEEQYRQLNQQHSQIKEIENQLRNVIQESTENSLSSKPRAPRTTPFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRLYSIKSTKMLIHPIDSESSE SEQ ID NO: 21 Equine ANGPTL3 241-455DFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCDVISGSSWILIQRRIDGSQNFNETWQNYKYGEGRLDFEFWLGLEKIYSIVKRSNYILRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVESTWDHKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKFRSKTKPERRRGICWKSQNGRLYTIKSTKML1HPID SEQ ID NO: 22 Equine ANGPTL3 225-455TTPSFHLNETKDVEHDDFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCDVISGSSWILIQRRIDGSQNFNETWQNYKYGFGRLDFEFWLGLEKIYSIVKRSNYILRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVFSTWDHKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKPRSKTKPERRRGICWKSQNGRLYTIKSTKMLIHPID SEQ ID NO: 23Equine ANGPTL3 207-455IQESTENSLSSKPRAPRTTPSFHLNETKDVEHDDFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCDVISGSSWILIQRRIDGSQNFNETWQNYKYGPGRLDFEFWLGLEKIYSIVKRSNYILRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVFSTWDHKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKPRSKTKPERRRGICWKSQNGRLYTIKSTKMLIHPIDSEQ ID NO: 24 Full length equine ANGPTL3 [equus caballus]MYTIKLFLVIAPLVISSRIDQDYSSLDSIPPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYALSLQTNEIKEEEKELRRTTSKLQVKNEEVKNMSLELNSKLESLLEEKSLLQQKVKYLEEQLTKLIKNQPEIQEHPEVTSLKTFVEQQDNSIKDLLQTMEEQYRQLNQQHSQIKEIENQLRRTGIQESTENSLSSKPRAPRTTPSEHLNETKDVEHDDFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCDVISGSSWILIQRRIDGSQNFNETWQNYKYGFGRLDFEFWLGLEKIYSIVKRSNYILRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVFSTWDHKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKPRSKTKPERRRGICWKSQNGRLYTIKSTKMLIHPIDSESFELRQIKKPMN SEQ ID NO: 25 241-455 consensusXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWXLIQXRXDGSQXFNETWXNYXXGFGRLDXEFWLGLEKIYKIVXXSNYXLEXELXDWXD(X)KXXXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCXEXXSGGWWXXXXCGXXNXNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX SEQ ID NO: 26 225-455 consensusTTPXXXXNEXXXXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWXLIQXRXDGSQXFNETWXNYXXGFGRLDXEFWLGLEKIYXIVXXSNYXLEXELXDWXD(X)KXXXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCXEXXSGGWWXXXXCGXXNXNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX SEQ ID NO: 27207-455 consensusXIXEXXEXSLSSKXRAPRTTPXXXXNEXXXXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWXLIQXRXDGSQXFNETWXNYXXGFGRLDXEFWLGLEKIYXIVXXSNYXLEXELXDWXD(X)KXXXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCXEXXSGGWWXXXXCGXXNXNGKYNKXXXKXXPERXX0XXWXXQXXXLYXIKXXKMXXX PXXSEQ ID NO: 28 Full length consensusMXTIKLXLXXXPLVIXSXXDXDXXSXDSXXXEPKSRFAMLDDVKILANGLLQLGHGLKDEVHKTKGQINDIFQKLNIFDQSFYXLSLXTXEIKEEEKELRRXTXXLQVKNEEVKNMSXELXSKXESLLEEKXXLQXKVXXLEXQLTXLIXXXXXXXEXXEVTSLKXXVEXQDNSIXXLLQXXEXQYXQLXQQXXQIKEIEXQLR(X)XXIXEXXEXSLSSKXRAPRTTPXXXXNEXXXXXXXXXPAXCXXXYNXGXHTSGXYXIXPXNSQXFXVYCDXXSGSXWXLIQXRXDGSQXFNETWXNYXXGFGRLDXEFWLGLEKIYXIVXXSNYXLEXELXDWXD(X)KXXXEYXFXLGXHETNYTLHXXXIXGNXXXAXPEXXDLXFSTWXHXAKGXXXCXEXXSGGWWXXXXCGXXNXNGKYNKXXXKXXPERXXGXXWXXQXXXLYXIKXXKMXXXPXX(SEXXEXXXXXXXXX) X = any amino acid Amino acidsin parentheses indicate that the amino acid can be absent or present

1. A pharmaceutical composition for intra-articular delivery, thecomposition comprising a pharmaceutically effective amount of apolypeptide comprising an amino acid sequence having at least 95%identity to SEQ ID NO:1 or
 25. 2. The composition of claim 1, whereinthe polypeptide is PEGylated.
 3. The composition of claim 1, wherein thepolypeptide is fused to a human serum albumin (HSA), an immunoglobulinheavy chain constant region (Fc), a polyhistidine, a glutathione Stransferase (GST), a thioredoxin, a protein A, a protein G, or a maltosebinding protein (MBP).
 4. The composition of claim 1, wherein thepolypeptide comprises 1, 2, 3, 4, or more unnatural amino acids.
 5. Thecomposition of claim 1, further comprising hyaluronic acid.
 6. Thecomposition of claim 1, wherein the polypeptide comprises SEQ ID NO:1 or25.
 7. The composition of claim 1, wherein the amino acid sequence hasat least 95% identity to SEQ ID NOs 2, 3, 4, 26, 27, or
 28. 8. Thecomposition of claim 1, wherein the amino acid sequence comprises SEQ IDNOs: 2, 3, 4, 26, 27, or
 28. 9. The composition of claim 1, wherein theamino acid sequence is at least 80% identical to SEQ ID NOs: 5, 9, 13,17, or
 21. 10. The composition of claim 1, wherein the amino acidsequence comprises SEQ ID NOs: 5, 9, 13, 17, or 21.